Project Summary/Abstract: Genomes acquire heritable and reversible chemical modifications that play a large role in influencing gene expression. These epigenetic modifications to the genome are fluid and often change allowing cells flexibility to alter patterns of gene expression based on environmental cues. 5- methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are epigenetic modifications of DNA associated with transcriptional regulation in vertebrate genomes. Consortium projects such as the ENCODE Project and the Roadmap Epigenomics Project have demonstrated unique patterning of epigenetically modified DNA in the genomes of diverse cell and tissue types alluding to a critical role in cell fate differentiation and disease progression. Within the retina, differentiation and maintenance of photoreceptors relies on precise transcriptional regulation of complex gene networks associated with unique patterns of DNA methylation. However, mechanistic detail of how DNA methylation and demethylation contribute to retinal development and disease remains unclear. Our preliminary evidence suggests a role for DNA methylation in directly modulating the interaction between retina-specific transcription factors and cis-regulatory elements in the mammalian retina. In this proposed study, we will use a combination of molecular, biochemical, and genomics analyses to dissect the functionality of DNA methylation on transcriptional regulation throughout the genome of the developing and diseased human retina. In Aim 1, we will employ quantitative gene-specific analysis of epigenetically modified DNA to test the hypothesis that photoreceptor-specific regulatory elements are actively demethylated during human retinal development. In Aim 2, we will biochemically characterize purified human CRX-DNA binding motif complexes to test the hypothesis that epigenetically modified DNAs dampen CRX's binding affinity. In Aim 3, we will employ genomics and bioinformatics methodologies to characterize global epigenetic regulation in the clinical context of age-related macular degeneration (AMD). The results of these proposed studies will not only fill a void in our understanding of photoreceptor development, but will also identify novel aspects of epigenetic gene regulatory mechanisms in the retina. !